Compositions and methods for modulating gamma-c-cytokine activity

ABSTRACT

The γc-family cytokines, Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin-9 (IL-9), Interleukin-15 (IL-15), and Interleukin-21 (IL-21), are associated with important human diseases, such as leukemia, autoimmune diseases, collagen diseases, diabetes mellitus, skin diseases, degenerative neuronal diseases and graft-versus-host disease (GvHD). Thus, inhibitors of γc-cytokine activity are valuable therapeutic and cosmetic agents as well as research tools. The present embodiments relate to the design of peptide antagonists based on the consensus γc-subunit binding site to inhibit γc-cytokine activity. In several embodiments, peptide antagonists exhibit Simul-Block activity, inhibiting the activity of multiple γc-cytokine family members.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2012/021566, filed Jan. 17, 2012, in the English language, whichclaims the benefit of U.S. Provisional Patent Application No.61/433,890, filed Jan. 18, 2011, and U.S. Provisional Patent ApplicationNo. 61/527,049, filed Aug. 24, 2011, the contents of each of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present embodiments relate to peptide antagonists of γc-familycytokines, a group of mammalian cytokines that are mainly produced byepithelial, stromal and immune cells and control the normal andpathological activation of a diverse array of lymphocytes. The presentembodiments also relate to the therapeutic uses of such peptides for thetreatment of certain human diseases. The present embodiments also relateto the cosmeceutical applications of such peptides. Description oftarget diseases, cosmeceutical applications, as well as methods ofadministration, production, and commercialization of the peptides aredisclosed.

BACKGROUND

Cytokines are a diverse group of soluble factors that mediate variouscell functions, such as, growth, functional differentiation, andpromotion or prevention of programmed cell death (apoptotic cell death).Cytokines, unlike hormones, are not produced by specialized glandulartissues, but can be produced by a wide variety of cell types, such asepithelial, stromal or immune cells.

More than 100 cytokines have been identified so far and are consideredto have developed by means of gene duplications from a pool ofprimordial genes (See Bazan, J. F. 1990, Immunol. Today 11:350-354). Insupport of this view, it is common for a group of cytokines to share acomponent in their multi-subunit receptor system. The mostwell-documented shared cytokine subunit in T cells is the common ysubunit (γc-subunit). The γc-subunit is shared by 6 known cytokines(Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-7 (IL-7),Interleukin-9 (IL-9), Interleukin-15 (IL-15), and Interleukin-21(IL-21), collectively called the “γc-cytokines” or “γc-familycytokines”) and plays an indispensable role in transducing cellactivation signals for all these cytokines. Additionally, for each ofthe γc-cytokines, there are one or two private cytokine-specificreceptor subunits that when complexed with the γc-subunit, give rise toa fully functional receptor. (See Rochman et al., 2009, Nat Rev Immunol.9: 480-90.)

The γc-family cytokines are a group of mammalian cytokines that aremainly produced by epithelial, stromal and immune cells and control thenormal and pathological activation of a diverse array of lymphocytes.These cytokines are critically required for the early development of Tcells in the thymus as well as their homeostasis in the periphery. Forexample, in the absence of the γc-subunit, T, B and NK cells do notdevelop in mice. (See Sugamura et al., 1996, Annu. Rev. Immunol.14:179-205).

Pathologies Associated with the γc-Cytokines

Recent studies have indicated that dysregulation of expression anddysfunction of the γc-cytokines could lead to a wide variety of humanimmunologic and hematopoietic diseases.

IL-2

While IL-2 was historically considered a prototype T cell growth factor,the generation of a knockout mouse lacking IL-2 expression revealed thatIL-2 is not critical for the growth or developmental of conventional Tcells in vivo. Over-expression of IL-2, however, leads to a preferentialexpansion of a subset of T-cells; the regulatory T cells (T-regs). (SeeAntony et al., 2006, J. Immunol. 176:5255-66.) T-regs suppress theimmune responses of other cells and thus act to maintain peripheraltolerance (reviewed in Sakaguchi et al., 2008, Cell 133:775-87).Breakdown of peripheral tolerance is thought to cause autoimmunediseases in humans. Thus, the immunosuppressive function of T-regs isthought to prevent the development of autoimmune diseases (See Sakaguchiet al., 2008, Cell 133:775-87). T-regs have also been implicated incancer, where solid tumors and hematologic malignancies have beenassociated with elevated numbers of T-regs (See De Rezende et al., 2010,Arch. Immunol. Ther. Exp. 58:179-190).

IL-4

IL-4 is a non-redundant cytokine involved in the differentiation of Thelper cells into the Th2 (T-helper type 2) subset, which promotes thedifferentiation of premature B cells into IgE producing plasma cells.IgE levels are elevated in allergic asthma. Thus, IL-4 is implicated inthe development of allergic Asthma. Antibodies targeting IL-4 can beused to treat or even prevent the onset of allergic asthma. (See LeBuanec et al., 2007, Vaccine 25:7206-16.)

IL-7

IL-7 is essential for B cell development and the early development of Tcells in the thymus. In mice, the abnormal expression of IL-7 causesT-cell-associated leukemia. (See Fisher et al., 1993, Leukemia2:S66-68.) However, in humans, misregulation of IL-7 does not appear tocause T-cell-associated leukemia. In humans, up-regulation of IL-7either alone or in combination with another γc-cytokine family member,IL-15, has been implicated in Large Granular Lymphocyte (LGL) leukemia.

IL-9

The role of IL-9 is still rather uncharacterized compared to otherγc-cytokine family members. Mice depleted of the IL-9 gene appear normaland do not lack any subsets of cells in the lymphoid and hematopoieticcompartments. Recent studies, however, reveal an in vivo role for IL-9in the generation of Th17 (T-helper induced by interleukin-17) cells(See Littman et al., 2010, Cell 140(6):845-58; and Nowak et al., 2009,J. Exp. Med. 206: 1653-60).

IL-15

IL-15 is critically involved in the development of NK cells, NK-T cells,some subsets of intraepithelial lymphocytes (IELs), γδ-T cells, andmemory-phenotype CD8 T-cells (See Waldmann, 2007, J. Clin. Immunol.27:1-18; and Tagaya et al., 1996, EMBO J. 15:4928-39.) Over-expressionof IL-15 in mice leads to the development of NK-T cell and CD8 cell typeT cell leukemia (See Fehniger et al., 2001, J. Exp. Med. 193:219-31;Sato et al. 2011 Blood in press). These experimentally induced leukemiasappear similar to LGL (large-granular lymphocyte) leukemia in humans,since in both instances the leukemic cells express CD8 antigen.

It is also suspected that IL-15-mediated autocrine mechanisms may beinvolved in the leukemic transformation of CD4 T lymphocytes. (See Azimiet al., 1998, Proc. Natl. Acad. Sci. 95:2452-7; Azimi et al., 1999, J.Immunol. 163:4064-72; Azimi et al., 2000, AIDS Res. Hum. Retroviruses16:1717-22; and Azimi et al., 2001, Proc. Natl. Acad. Sci. 98:14559-64).For example, CD4-tropic HTLV-I, which causes Adult T cell leukemia inhumans, induces autocrine growth of virus-transformed T cells throughthe production of IL-15 and IL-15Rα (Azimi et al., 1998, Proc. Natl.Acad. Sci. 95:2452-7).

In addition to leukemic transformation, recent studies implicate IL-15in the pathological development of Celiac disease (CD), an autoimmunedisease. IL-15 is known to stimulate the differentiation of NK, CD8 andintestinal intraepithelial lymphocyte (IEL) cells intolymphokine-activated killer (LAK) cells by inducing the expression ofcytolytic enzymes (i.e., Granzyme and Perforin) as well as interferon-γ.Celiac Disease (denoted CD from herein) is an immune-mediatedenteropathy that is triggered by the consumption of gluten-containingfood in individuals that express specific HLA-DQ alleles. The prevalenceof this disease is 1% in the western population. The only currenttreatment for CD is the complete elimination of gluten from thepatient's diet. The pathology of CD is mainly caused by extensive damageto the intestinal mucosa, which is caused by activated CD8 T cells thathave infiltrated to the intestinal lamina propria. These CD8 T cellsappear to be activated through mechanisms involving IL-15. One recentpublication demonstrated in mice that ectopic over-expression of IL-15by enterocytes leads to the development of enteropathy, which closelyresembles the lesions in CD patients. Neutralization of IL-15 activitydramatically diminished the pathological changes. Thus, an interventionblocking the activation of CD8 T cells by IL-15 appears to provide analternative strategy in managing CD to the conventional gluten-freediet.

IL-21

IL-21 is the most recently discovered member of the γc-family. Unlikeother family members, IL-21 does not appear to have potentgrowth-promoting effects. Instead, IL-21 is thought to function more asa differentiation factor than a factor controlling cellularproliferation (See Tagaya, 2010, J. Leuk. Biol. 87:13-15).

Current Strategies for Treating γc-Cytokine-Mediated Disorders

Because the γc-cytokines are thought to be involved in numerous humandiseases, several methods of treating γc-cytokine-implicated diseases byinhibiting γc-cytokine family activities have been proposed. Thesemethods include the use of cytokine-specific monoclonal antibodies toneutralize the targeted cytokine's activity in vivo; use of monoclonalantibodies targeting the private cytokine-specific receptor subunits(subunits other than the shared γc-subunit) to selectively inhibitcytokine activity; and use of chemical inhibitors that block thedownstream intracellular cytokine signal transduction pathway. Whilecytokine-specific antibodies are often the first choice in designingtherapeutics, cytokines that share receptor components displayoverlapping functions (See Paul, W. E., 1989, Cell 57:521-24) and morethan one cytokine can co-operate to cause a disease (see exampledescribed below). Thus, approaches involving neutralization of a singlecytokine may not be effective in the treatment of cytokine-implicatedhuman diseases.

Strategies for designing therapeutics that inhibit the function ofmultiple cytokines via antibodies which recognize a shared receptorcomponent have also been proposed. However, the multi-subunit nature ofcytokine receptor systems and the fact that functional receptors for asingle cytokine can assume different configurations makes this approachdifficult. For example, a functional IL-15 receptor can be eitherIL-15Rβ/γc or IL-15Rα/β/γc. (See Dubois et al., 2002, Immunity17:537-47.) An antibody against the IL-15Rβ receptor (TMIβ1), is anefficient inhibitor of the IL-15 function, but only when the IL-15Rαmolecule is absent from the receptor complex. (See Tanaka et al., 1991,J. Immunol. 147:2222-28.) Thus, the effectiveness of a monoclonalanti-receptor antibody, whether raised against a shared or a privatesubunit, can be context-dependent and is unpredictable in vivo.

Although clinical use of monoclonal antibodies against biologicallyactive factors or receptors associated with the pathogenesis of diseasesis an established practice, there are few demonstrations of successfuloutcomes. Moreover, establishment of a clinically-suited monoclonalantibody treatment is a long and difficult process, with the successfulgeneration of a neutralizing antibody largely a matter of luck. Forexample, due to the critical importance of the γc-subunit in mediatingsignaling by γc-family cytokines, many attempts to generate polyclonaland monoclonal antibodies against the γc-subunit have been made andthere exist many commercial antibodies recognizing the γc-subunit inmice and in humans. Curiously, however, none of these anti-γc-subunitantibodies block the function of the γc-cytokines.

Another problem with the therapeutic use of monoclonal antibodies isthat monoclonal antibodies are usually generated by immunizing rodentswith human proteins, so the generated antibody is a foreign protein andthus highly immunogenic. To circumvent this problem, the amino acidsequence of the monoclonal antibody is molecularly modified so that theantibody molecule is recognized as a human immunoglobulin (a processcalled humanization), but this process requires time and expense.

Targeting JAK3, as an Existing Alternative Example for the Inhibition ofMultiple γc-cytokines

The interaction between the γc-subunit and a γc-cytokine leads to theactivation of an intracellular protein tyrosine kinase called Januskinase 3 (Jak3). Jak3, in turn, phosphorylates multiple signalingmolecules including STATS, and PI3 kinase. The interaction of theγc-subunit and Jak3 is very specific. In fact, there is no otherreceptor molecule that recruits Jak3 for signal transduction. (SeeO'Shea, 2004, Ann. Rheum. Dis. 63:(suppl. II):ii67-7.) Thus, theinhibition of cytokine signaling through the γc-subunit can beaccomplished by blocking the activity of Jak3 kinase. Accordingly,multiple chemical inhibitors that target the kinase activity of Jak3have been introduced to the market. (See Pesu et al., 2008, Immunol.Rev. 223:132-142.) One such example is CP690,550.

The major shortcoming of these protein kinase inhibitors is the lack ofspecificity to Jak3 kinase. These drugs intercept the binding of ATP(adenosine-triphosphate) molecules to Jak3 kinase, a common biochemicalreaction for many protein kinases, and thus tend to block the action ofmultiple intracellular protein kinases that are unrelated to Jak3 kinasewhose actions are critically needed for the well-being of normal cellsin various tissues. Thus, more specific inhibitors of signaling throughthe γc-subunit are needed.

There is therefore a great need for an alternative strategy for treatingγc-cytokine-implicated diseases.

SUMMARY OF THE INVENTION

One embodiment relates to an isolated or purified peptide, consistingessentially of a 19-mer amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) (referred to hereinas “BNZ-γ” (BNZ-gamma)).

Another embodiment relates to a method for blocking signaling by one ormore γc-cytokine family members, comprising contacting a cell with anisolated or purified peptide consisting essentially of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1).

Another embodiment relates to a method for blocking signaling by one ormore γc-cytokine family members, comprising contacting a cell with anisolated or purified peptide consisting essentially of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), whereinthe cell is an immune cell.

Another embodiment relates to a method for blocking signaling by one ormore γc-cytokine family members, comprising contacting a cell with anisolated or purified peptide consisting essentially of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), whereinthe γc-cytokine family member is selected from the group consisting of:IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21.

Another embodiment relates to derivative peptides of a peptideconsisting of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1), wherein the derivative peptide has similarphysico-chemical properties as the peptide consisting of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but thederivative peptide has distinct biological activity.

Another embodiment relates to a custom peptide wherein the amino acidsequence of the custom peptide differs from amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) by conservativesubstitution of one or more amino acids.

Another embodiment relates to a custom peptide, consisting essentiallyof a 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1).

Another embodiment relates to a custom peptide wherein the amino acidsequence of the custom peptide differs from amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) by substitutinganother polar amino acid for the glutamine (Q) at the 6-position.

Another embodiment relates to a custom peptide wherein the amino acidsequence of the custom peptide differs from the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) by substitution ofone or more amino acids with similar biochemical properties to the aminoacids comprising sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ IDNO: 1).

Another embodiment relates to custom peptide derivatives of the aminoacid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S, wherein the aminoacid sequence of the custom peptide has similar physico-chemicalproperties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, wherein the amino acid sequence of the custompeptide shares at least 50% sequence homology to the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1).

Another embodiment relates to a conjugation of a peptide consistingessentially of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1) to the N-termini, C-termini and/or to the side residuesof existing biological proteins/peptides for efficient delivery andimproved biological stability in vivo. Examples of such conjugations areBSA, albumin, Fc region of IgG, other biological proteins that functionas scaffold, Poly Ethylene Glycol or (PEG) at different molecularweights or other similar moieties.

Another embodiment relates to conjugation of custom peptide derivativesof the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ IDNO: 1) to the N-termini, C-termini and/or to the side residues ofexisting biological proteins/peptides, wherein the amino acid sequenceof the custom peptide has similar physico-chemical properties as apeptide of the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1), but has distinct biological activity, wherein the aminoacid sequence of the custom peptide shares at least 50% sequencehomology to the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). Examples of suchconjugations are albumin, Fc region of IgG, other biological proteinsthat function as scaffold, or Poly Ethylene Glycol or (PEG) at differentmolecular weights or other similar moieties.

Another embodiment relates a method of inhibiting γc-cytokine activitycomprising, contacting a γc-cytokine with a peptide consistingessentially of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1).

Another embodiment relates to polyclonal and monoclonal antibodiesraised against an immunogenic peptide comprising amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1).

Another embodiment relates to polyclonal and monoclonal antibodiesraised against custom peptide derivatives of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), wherein the aminoacid sequence of the custom peptide has similar physico-chemicalproperties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, and wherein the amino acid sequence of the custompeptide shares at least 50% sequence homology to the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an alignment of the D-helix region of human γc-cytokinefamily members.

FIG. 1B depicts the γc-box and IL-2/IL-15 box motifs which give rise tothe consensus sequence around the D-helix region of the γc-cytokines.

FIG. 2 depicts a diagramed representation of the biochemical propertiesof amino acids.

FIG. 3A shows inhibition of IL-2, IL-15, and IL-9 activity by BNZ-γ in aPT-18 proliferation assay.

FIG. 3B shows a proliferation assay of CTTL2 cells grown in the presenceof IL-2 or IL-15 and 0, 0.1, 1 or 10 uM BNZ-65 .

FIG. 3C shows inhibition of IL-15-mediated tyrosine-phosphorylation ofSTATS by BNZ-γ.

FIG. 4A shows an ex vivo T-cell proliferation assay using HAM/TSPperipheral blood. T-cell proliferation is inhibited by addition ofBNZ-γ.

FIG. 4B shows the population of CD4+CD25+cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is diminished afteradding BNZ-γ to the culture.

FIG. 4C shows the population of CD4+Ki67 cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is reduced afteradding BNZ-γ to the culture.

FIG. 4D shows the percent of live cells by Guava staining in an ex vivoT-cell proliferation assay using HAM/TSP peripheral blood is notimpacted after adding BNZ-γ to the culture.

DETAILED DESCRIPTION Overview

The γc-cytokines are important players in the development of thelymphoid cells that constitute the immune system, particularly T, B, andNK cells. Further, γc-cytokines have been implicated in various humandiseases. Thus, factors that inhibit γc-cytokine activity would provideuseful tools to elucidate the developmental mechanism of subsets oflymphocytes and to treat immune disorders and γc-cytokine-mediateddiseases.

Germ line depletion of the genes encoding the γc-subunit in mice ormutations of γc-subunit in humans are known to cause severe combineimmunodeficiency (SCID) by disrupting the normal appearance or functionof NK, T, and B cells. The importance of the γc-subunit in the signaltransduction of the γc-cytokines, IL-2, -4, -7, -9, 15, -21, isindicated in studies demonstrating the a of response of lymphocytes fromthese mice and human patients to the γc-cytokines (reviewed in Sugamuraet al., 1995 Adv. Immunol. 59:225-277). This indicates that disruptionof the interaction between the γc-subunit and a γc-cytokine wouldefficiently block the intracellular signaling events by the γc-cytokinefamily members. Therefore antagonist peptides according to the presentembodiments are expected to effectively block the pathogenic changes inhumans suffering from the diseases mediated by misregulation of theγc-cytokine family members.

As an alternative to antibody-mediated approaches for modulating theactivity of individual γc-cytokines, Applicants have devised novel, lowmolecular weight compounds herein referred to as “Simul-Block”, whichsuppress the activity of multiple γc-cytokines. These low molecularweight compounds, which include both chemicals and peptides, are lessimmunogenic than antibodies. These properties distinguish Simul-Block asa more efficient strategy for mediating γc-cytokine activity in clinicalinterventions.

Discovery of the γc-Box

The C-terminus (the D-helix) of the γc-cytokines contains the proposedsite for interacting with the common γc-subunit of the multi-unitcytokine receptors. (Bernard et al., 2004 J. Biol. Chem. 279:24313-21.)Comparison of the biochemical properties of the amino acids of allγc-cytokines identified in mice and humans revealed that the chemicalnature of the amino acids, for example, hydrophobicity, hydrophilicity,base/acidic nature, are conserved, if not identical, at many positionsin the D-helix across the members of the γc-cytokine family. Incontrast, the sequence of IL-13, which is related to the γc-cytokine,IL-4, but does not bind to the γc-subunit, does not exhibit significanthomology in the D-helix region to the γc-cytokines, suggesting that thesequence homology in the D-helix region is correlated with binding tothe γc-subunit. As shown in FIG. 1, alignment of the amino acidsequences of the D-helix region of γc-cytokine family members in humansreveals a motif of moderate sequence homology in these cytokinesreferred to herein as “the γc-box”.

The γc-box comprises 19 amino acids where out of the 19 positions,positions 4, 5, and 13 are fully conserved as Phenylalanine, Leucine,and Asparagine, respectively. Less conservation is observed at positions6, 7 and 11 of the γc-box where the amino acid is one of two or threerelated amino acids that share physico-chemical properties: position 6may be occupied by the polar amino acids Glutamate, Asparagine orGlutamine; non-polar amino acids Serine or Arginine can occupy position7; and position 11 is occupied by either of the non-polar aliphaticamino acids Leucine or Isoleucine. Positions 9 and 16 may be occupied bythe either the non-polar amino acid Isoleucine or the polar amino acidLysine. See FIG. 1B. Some differences in the amino acid composition ofthe γc-box are observed at positions 9 and 6 amongst subfamilies of theγc-cytokines. Comparison of the γc-cytokines across species indicatesthat Isoleucine is present at the 9 and 6 positions in the IL-2/15subfamily, whereas the other γc-family members (e.g., IL-4, IL-21)possess Lysine in these positions. Not wishing to be bound by aparticular theory, Isoleucine and Lysine are biochemically different andthus may impart specific conformational differences between the IL-2/15subfamily and other γc-cytokines.

Conservation of the γc-box motif between γc-cytokines is supported byfindings that an Asparagine (Asn, Q) residue located in the D-helixregion is critical for the binding of the γc-cytokines to theγc-subunit. (Bernard et al., 2004 J. Biol. Chem. 279: 24313-21.)

Peptide Inhibitors of γc-Cytokine Activity

The activity of γc-family cytokines may be blocked by disrupting theinteraction between the γc-cytokine and the γc-subunit, for example byintroducing a competitive inhibitor which can interact with theγc-subunit without stimulating signaling through the multi-subunitcytokine receptors. Not to be bound by a particular theory, theconserved γc-box motif, which participates in binding of the γc-familycytokines to the γc-subunit, presents a core base amino acid sequencewhich can be utilized to design peptide inhibitors of γc-cytokinesignaling.

The core γc-box amino acid sequence comprises:D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid). Embodiments described herein relate to custom peptidederivatives of the core γc-box amino acid sequence which can inhibit theactivity of one or more γc-cytokines. Custom peptide derivatives includeany peptide whose partial amino acid sequence shows approximately 50%,50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identityto the core γc-box amino acid sequence. Custom peptide derivativesfurther include any peptide wherein a partial amino acid sequence ofthat peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of the core γc-box. Forexample, amino acids with similar physico-chemical properties wouldinclude Phenylalanine, Tyrosine, Tryptophan, and Histidine, which arearomatic amino acids. FIG. 2 shows a diagrammed representation of aminoacids with similar physico-chemical properties which may be may besubstituted for the amino acids comprising the core γc-box. Peptidederivatives of the core γc-box may be 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50amino acids in length. In some embodiments, the custom peptidederivatives may be conjugated to the N-termini, C-termini and/or to theside residues of existing biological proteins/peptides.

Based on the identification of the conserved γc-box motif in cytokineswhich bind to the γc-subunit, Applicants have devised a novel, 19-mercustom derivative peptide which is an artificial composite peptidecombining the amino acid sequence of the human IL-2 and IL-15 γc-box.The 19-mer peptide, herein referred to as BNZ-γ, consists of the aminoacid sequence: I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1),where the amino acids depicted by bold characters are conserved betweenIL-2 and IL-15 and the underlined amino acids represent positions wherethe physico-chemical properties of the amino acids are conserved.

Applicants discovered that the 19-mer BNZ-γ, suppresses IL-15 and IL-19induced cellular proliferation, but not IL-2 or IL-4 induced cellularproliferation. See FIG. 3A and Example 2. Applicants furtherdemonstrated that BNZ-γ inhibits IL-15 mediated phosphorylation of theintracellular cytokine signal transduction molecule, STAT-5. See FIG. 3Cand Example 5. These results demonstrate that custom peptide derivativesof the conserved γc-box motif can inhibit the activity of multipleγc-cytokines.

Several embodiments relate to custom derivative peptides of the 19-merBNZ-γ amino acid sequence, I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ IDNO: 1), which can inhibit the activity of one or more γc-cytokines.Custom peptide derivatives of the 19-mer BNZ-γ amino acid sequenceinclude any peptide whose partial amino acid sequence showsapproximately 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity to amino acid sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). Custom peptidederivatives further include any peptide wherein a partial amino acidsequence of that peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). In severalembodiments, the amino acid residues of the custom derivative peptidesretain similar physico-chemical properties with the amino acid residuesof BNZ-γ, but exhibit different biological inhibition specificity to the6 γc-cytokine family members from that of the original 19-mer peptide.Peptide derivatives of BNZ-γ may be 19, 20, 21, 22, 24, 25-30, 30-35,35-40, 40-45, 45-50, or more than 50 amino acids in length. In someembodiments, the custom peptide derivatives may be conjugated to theN-termini, C-termini and/or to the side residues of existing biologicalproteins/peptides.

Several embodiments relate to custom peptide derivatives of the γc-boxmotifs of IL-5, IL-2, IL-21, IL-4, IL-9, or IL-7, which are depicted inFIG. 1A. Other embodiments relate to custom derivative peptides whichare artificial composite peptides combining the amino acid sequence oftwo or more of the human IL-5, IL-2, IL-21, IL-4, IL-9, and IL-7 γc-boxmotifs. Several embodiments relate to custom peptide derivatives of theof the γc-box motifs of IL-5, IL-2, IL-21, IL-4, IL-9, or IL-7 having apartial amino acid sequence that shows approximately 50%, 50-60%,60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to aminoacid sequences of the of the γc-box motifs of IL-5, IL-2, IL-21, IL-4,IL-9, or IL-7. Custom peptide derivatives of the of the γc-box motifs ofIL-5, IL-2, IL-21, IL-4, IL-9, or IL-7 further include any peptidewherein a partial amino acid sequence of that peptide derivativecomprises amino acids with similar physico-chemical properties to theamino acids of sequence of the γc-box motifs of IL-5, IL-2, IL-21, IL-4,IL-9, or IL-7.

Several embodiments relate to custom peptide derivatives that wouldinhibit the function of one, all, or selective members of theγc-cytokines. In some embodiments, the custom peptide derivativesselectively target individual γc-cytokine family members. For example, acustom peptide derivative can selectively inhibit the function of IL-2,IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, a custompeptide derivative can inhibit 2 or more γc-cytokine family members. Forexample, the custom peptide derivatives of the present embodiments canselectively inhibit the function of IL-2 in combination with one or moreof IL-4, IL-7, IL-9, IL-15, and IL-21; IL-4 in combination with one ormore of IL-7, IL-9, IL-15, and IL-21; IL-7 in combination with one ormore of IL-9, IL-15, and IL-21; IL-9 in combination with one or more ofIL-2, IL-4, IL-7, IL-15, and IL-21; IL-15 in combination with one ormore of IL-2, IL-4, IL-7, IL-9, and IL-21; or IL-21 in combination withone or more of IL-2, IL-4, IL-7, IL-9, and IL-15. In other embodiments,custom peptide derivatives can comprehensively target all γc-cytokinefamily members. Not wishing to be bound by a particular theory, thecustom peptide derivatives can inhibit the function of all or selectivemembers of the γc-cytokines by diminishing the binding of γc-cytokinesto the γc-subunit, for example, as a competitive inhibitor. Such custompeptide derivatives may be used in diverse applications, including as aclinical drug.

The terms “oligopeptide,” “polypeptide,” “peptide,” and “protein” can beused interchangeably when referring to the custom peptide derivativesprovided in accordance with the present embodiments and can be used todesignate a series of amino acid residues of any length. Peptidesaccording to the present embodiments may also contain non-natural aminoacids. Linker elements can be joined to the peptides of the presentembodiments through peptide bonds or via chemical bonds. The peptides ofthe present embodiments may be linear or cyclic, and may include (D) aswell as (L) amino acids. Peptides of the present embodiments may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylamino), with or without any of a wide variety of sidechain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylamino, and thelike). Residues other than common amino acids that may be presentinclude, but are not limited to, penicillamine, tetramethylene cysteine,pentamethylene cysteine, mercaptopropionic acid,pentamethylene-mercaptopropionic acid, 2-mercaptobenzene,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,aminoadipic acid, m-aminomethylbenzoic acid, and diaminopropionic acid.

Peptides of the present embodiments can be produced and obtained byvarious methods known to those skilled in the art. For example, thepeptide may be produced by genetic engineering, based on the nucleotidesequence coding for the peptide of the present embodiments, orchemically synthesized by means of peptide solid-phase synthesis and thelike, or produced and obtained in their combination. One skilled in theart can synthesize the custom peptide derivatives based on the presentdisclosure of the conserved γc-box motif and knowledge of thebiochemical properties of amino acids as described in FIG. 2. Someembodiments also relate to polynucleotides comprising nucleotidesequences encoding the peptides of the present invention. “Nucleotidesequence,” “polynucleotide,” or “nucleic acid” can be usedinterchangeably, and are understood to mean either double-stranded DNA,a single-stranded DNA or products of transcription of the said DNAs(e.g., RNA molecules). Polynucleotides can be administered to cells orsubjects and expressed by the cells or subjects, rather thanadministering the peptides themselves. Several embodiments also relateto genetic constructs comprising a polynucleotide sequence encoding thepeptides of the present invention. Genetic constructs can also containadditional regulatory elements such as promoters and enhancers and,optionally, selectable markers.

Methods of Treating γc-Cytokine Mediated Diseases

Several embodiments relate to the use of γc-antagonist peptides in thetreatment of γc-cytokine mediated diseases. Use of custom peptidederivative according to the present embodiments allows for flexibilityin the design of the therapeutic agent (custom design of the peptide)and enables more comprehensive outcomes, which would not be accomplishedby conventional strategies employing anti-cytokine or anti-cytokinereceptor antibodies.

Described herein is a novel method of blocking the action of γc-familycytokines. Such manipulations can yield effective methods of clinicalinterventions in treating diseases related to the dysregulation ordysfunction of γc-cytokines. Examples of disease that may be treated bydisrupting the interaction between the γc-cytokine and the γc-subunitinclude autoimmune diseases such as systemic lupus erythematosis,Sjoegren's syndrome, Wegener's granulomatosis Celiac disease,Hashimoto's or auto-immune thyroiditis; collagen diseases includingrheumatoid arthritis, inflammatory bowel disease, diabetes mellitus,autoimmune diseases of the skin such as psoriasis; degenerative neuronaldiseases such as multiple sclerosis, uvietis or inflammation of the eyeand sympathetic ophthalmia, graft-versus-host disease (GvHD) andmyasthenia gravis.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of 1-Human T-cell Lymphotropic type I and II(HTLV-I and HTLV-II)-associated diseases including Adult T-cell Leukemia(ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy and myositis. In some embodiments, the γc-antagonistpeptides described herein may be used in the treatment of other viraldiseases such as influenza, AIDS, HBV and Herpes or parasitic diseases.

In several embodiments, the γc-antagonist peptides may be administeredbefore, during, and or after transplantation of various organs as animmunosuppressant agent.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of immune-mediated diseases such as asthma andother inflammatory respiratory diseases, such as, but not limited tosinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease(COPD), allergic rhinitis, acute and chronic otitis, lung fibrosis. Insome embodiments, γc-antagonist peptides may be administered to treat orprevent allergic reactions due to exposure to allergens, chemical agentsor other common causes of acute respiratory disease. In someembodiments, γc-antagonist peptides may be administered to treat orprevent inflammatory responses caused by viruses, bacteria, chemicalreagents, and biochemical reagents.

In several embodiments, the γc-antagonist peptides may be administeredto treat some types of malignancies such as LGL-leukemia,Intraepithelial lymphoma and leukemia in Refractory Celiac Disease, NKleukemia/lymphoma and NK-T leukemia/lymphoma

In some embodiments, custom peptide derivatives according to theembodiments described herein can be used for cosmetic purposes, such asthe treatment of acne, hair loss, sunburn and nail maintenance, includedto ointment as anti-aging component because of the anti-inflammatorynature of them.

Several embodiments relate to therapeutic antagonist peptides that wouldinhibit the function of all or selective members of the γc-cytokines. Insome embodiments, therapeutic antagonist peptides selectively inhibitindividual γc-cytokine family members (custom peptides). In otherembodiments, therapeutic antagonist peptides can comprehensively inhibitall γc-cytokine family members (Simul-Block). In some embodiments,therapeutic antagonist peptides selectively inhibit subsets of theγc-cytokines. Not wishing to be bound by a particular theory, thepeptide antagonists can inhibit the function of all or selective membersof the γc-cytokines by diminishing the binding of γc-cytokines to theγc-subunit, for example, as a competitive inhibitor.

Several members of the γc-cytokine family, IL-2, IL-7, and IL-15, butnot IL-4 have been implicated as being involved in graft versus hostdisease (GvHD) in an experimental mouse model. (Miyagawa et al., 2008 J.Immunol. 181:1109-19.) One embodiment relates to the use of therapeuticantagonist peptides that selectively inhibit IL-2, IL-7, and IL-15activity for the treatment of GvHD in humans, allowing survival of thegrafted tissues or bone marrow cells. Other embodiments relate to theuse of therapeutic antagonist peptides that selectively inhibit acombination of IL-2 and IL-7, IL-2, and IL-15, or IL-7 and IL-15 totreat GvHD. Other embodiments relate to the use of a combination oftherapeutic antagonist peptides that selectively inhibit IL-2, IL-7, orIL-15.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-2 function for the treatment of autoimmunedisorders where T-regs have been implicated as playing a role. In someembodiments, peptide-mediated inhibition of T-regs can enhance thenatural anti-cancer immunity in humans, providing a novel means ofanti-cancer therapy.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-4 to treat asthma.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-7 either alone or in combination withtherapeutic antagonist peptides that selectively inhibit the γc-cytokinefamily member, IL-15, as a therapeutic agent for LGL leukemia. In someembodiments therapeutic antagonist peptides that selectively inhibitboth IL-7 and IL-15 activity can be used to treat LGL leukemia. Severalembodiments relate to the use of BNZ-γ to treat LGL leukemia. In someembodiments, specific γc-antagonist peptides that selectively IL-15alone or specific γc-antagonist peptides that selectively IL-15 and IL-7are used as a therapeutic agent for CD4/CD8 T lymphocyte-associatedleukemia including that caused by the HTLV-I.

Several embodiments relate to the use of γc-antagonist peptides thatselectively inhibit the activity of IL-9, either alone or in combinationwith the other γc-cytokine family members, as a therapeutic agent forhuman diseases that involve the abnormal development of Th17 cells.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-15 activity as a therapeutic agent fortreating CD. One recent publication suggested that IL-21, in addition toIL-15, may play a role in CD pathogenesis. (See Bodd et al., 2010,Mucosal Immunol. 3:594-601.) This suggests that optimum treatment of CDby conventional anti-cytokine or cytokine-receptor antibodies wouldbenefit from a combination of at least two antibodies recognizingcomponent that belong to the IL-15 and IL-21 systems. In someembodiments, custom derivative antagonist peptides that selectivelyinhibit both IL-15 and IL-21 activity are used as a therapeutic agentfor treating CD.

In addition to having therapeutic applications, γc-antagonist peptideshave applications in consumer products as well. Several embodimentsrelate to the use of γc-antagonist peptides in skin care products suchas anti-aging, anti-inflammatory, anti-acne, and other relatedapplications. Some embodiments relate to the use of γc-antagonistpeptides in hair products as anti-hair loss ingredient to treat hairloss caused by autoimmune disorders.

Another embodiment relates to the development of chemical compounds(non-peptide, non-protein) that have a spatial structure which resemblesthe 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1) and can fit into the pocket of the γc-subunit tostructurally hinder the access of a γc-cytokine to the γc-subunit forbinding. Some embodiments relate to the use of structurally similarchemical compounds as inhibitors of γc-cytokine activity. Such molecularmimicry strategy to further refine the development of syntheticcompounds resembling in structure to existing biologicalpeptide/proteins is described in Orzaez et al., 2009 Chem. Med. Chem.4:146-160. Another embodiment relates to administration of chemicalcompounds (non-peptide, non-protein) that have a resembling 3D structureas the 19-mer amino acids sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1) to treat γc-cytokine-mediated diseases.

Several embodiments relates to the administration of a peptide of aminoacid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) totreat γc-cytokine-mediated diseases. Another embodiment relates to theadministration of derivative peptides of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), wherein the aminoacid sequence of the derivative peptide has similar physico-chemicalproperties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of a peptide of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) conjugated to theN- and C-termini or to the side residues of existing biologicalproteins/peptides into patients to treat γc-cytokine-mediated diseases.

Several embodiments relate to administration of polyclonal andmonoclonal antibodies raised against a peptide comprising of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) intopatients as an immunogen to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of polyclonal and monoclonalantibodies that were raised against derivative peptides of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) whereinthe amino acid sequence of the derivative peptide has similarphysico-chemical properties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, into patients as an immunogen to treatγc-cytokine-mediated diseases.

Administration of γc-Antagonist Peptides

The present embodiments also encompass the use of γc-antagonist peptidesfor the manufacture of a medicament for the treatment of a disease. Thepresent embodiments also encompass a pharmaceutical composition thatincludes γc-antagonist peptides in combination with a pharmaceuticallyacceptable carrier. The pharmaceutical composition can include apharmaceutically acceptable carrier and a non-toxic therapeuticallyeffective amount of γc-antagonist peptides, or other compositions of thepresent embodiments.

The present embodiments provide methods of using pharmaceuticalcompositions comprising an effective amount of antagonists forγc-cytokines in a suitable diluent or carrier. A γc-antagonist of thepresent embodiments can be formulated according to known methods used toprepare pharmaceutically useful compositions. A γc-antagonist can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,phosphate, acetate, Tris-HCl), preservatives (e.g., thimerosal, benzylalcohol, parabens), emulsifying compounds, solubilizers, adjuvants,and/or carriers such as bovine serum albumin. Suitable carriers andtheir formulations are described in Remington's Pharmaceutical Sciences,16^(th) ed. 1980 Mack Publishing CO. Additionally, such compositions cancontain a γc-antagonist complexed with polyethylene glycol (PEG), metalions, or incorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroblasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance or a γc-antagonist. A γc-antagonist can beconjugated to antibodies against cell-specific antigens, receptors,ligands, or coupled to ligands for tissue-specific receptors.

Methods of administrating γc-antagonists of the present embodiments maybe selected as appropriate, depending on factors, such as the type ofdiseases, the condition of subjects, and/or the site to be targeted. Theγc-antagonists can be administered topically, orally, parenterally,rectally, or by inhalation. The term “parenteral” includes subcutaneousinjections, intravenous, intramuscular, intraperitoneal, intracisternalinjection, or infusion techniques. These compositions will typicallyinclude an effective amount of a γc-antagonist, alone or in combinationwith an effective amount of any other active material. The amount of thepeptide contained in pharmaceutical compositions of the presentembodiments, dosage form of the pharmaceutical compositions, frequencyof administration, and the like may be selected as appropriate,depending on factors, such as the type of diseases, the condition ofsubjects, and/or the site to be targeted. Such dosages and desired drugconcentrations contained in the compositions may vary affected by manyparameters, including the intended use, patient's body weight and age,and the route of administration. Pilot studies will first be conductedusing animal studies and the scaling to human administration will beperformed according to art-accepted practice.

In one embodiment, host cells that have been genetically modified with apolynucleotide encoding at least one γc-antagonist peptide areadministered to a subject to treat a proliferation disorder and/or toreduce the growth of malignant cells. The polynucleotide is expressed bythe host cells, thereby producing the peptides within the subject.Preferably, the host cells are allogeneic or autogeneic to the subject.

In a further aspect, γc-antagonist peptides can be used in combinationwith other therapies, for example, therapies inhibiting cancer cellproliferation and growth. The phrase “combination therapy” embraces theadministration of γc-antagonist peptides and an additional therapeuticagent as part of a specific treatment regimen intended to provide abeneficial effect from the co-action of these therapeutic agents.Administration of these therapeutic agents in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected).

A combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. There therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

Combination therapy also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different therapeutic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment). Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporarily removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-proliferative agent selected from thegroup consisting of chemotherapeutic agent, an antimetabolite, andantitumorgenic agent, and antimitotic agent, and antiviral agent, andantineoplastic agent, an immunotherapeutic agent, and a radiotherapeuticagent.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-inflammatory agent selected from thegroup consisting of steroids, corticosteroids, and nonsteroidalanti-inflammatory drugs.

Also provided are kits for performing any of the above methods. Kits mayinclude a γc-antagonist according to the present embodiments. In someembodiments, the kit may include instructions. Instructions may be inwritten or pictograph form, or may be on recorded media including audiotape, audio CD, video tape, DVD, CD-ROM, or the like. The kits maycomprise packaging.

Definitions

As used herein, the term “patient” refers to the recipient of atherapeutic treatment and includes all organisms within the kingdomanimalia. In preferred embodiments, the animal is within the family ofmammals, such as humans, bovine, ovine, porcine, feline, buffalo,canine, goat, equine, donkey, deer, and primates. The most preferredanimal is human.

As used herein, the term “treat” or any variation thereof (e.g.,treatment, treating, etc.), refers to any treatment of a patientdiagnosed with a biological condition, such as CD4-, CD8-, andLGL-leukemia, an autoimmune disease, systemic lupus erythematosis,Sjoegren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, a collagen disease, rheumatoid arthritis,inflammatory bowel disease, diabetes mellitus, psoriasis, a degenerativeneuronal disease, multiple sclerosis, uvietis, inflammation of the eye,graft-versus-host disease (GvHD), myasthenia gravis, 1-Human T-cellLymphotropic type I and II (HTLV-I and HTLV-II)-associated diseases,Adult T-cell Leukemia (ATL), HTLV-associated Myelopathy/Tropical SpasticParaparesis (HAM/TSP), uveitis (HU), arthropathy, pneumopathy,dermatitis, exocrinopathy, myositis, influenza, AIDS, HBV, Herpes,asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonarydisease (COPD), allergic rhinitis, acute and chronic otitis, lungfibrosis, NK leukemia/lymphoma and NK-T leukemia/lymphoma. The termtreat, as used herein, includes: (i) preventing or delaying thepresentation of symptoms associated with the biological condition ofinterest in an at-risk patient who has yet to display symptomsassociated with the biological condition; (ii) ameliorating the symptomsassociated with the biological condition of interest in a patientdiagnosed with the biological condition; (iii) preventing, delaying, orameliorating the presentation of symptoms associated with complications,conditions, or diseases associated with the biological condition ofinterest in either an at-risk patient or a patient diagnosed with thebiological condition; (iv) slowing, delaying or halting the progressionof the biological condition; and/or (v) preventing, delaying, slowing,halting or ameliorating the cellular events of inflammation.

The term “symptom(s)” as used herein, refers to common signs orindications that a patient is suffering from a specific condition ordisease.

The term “effective amount,” as used herein, refers to the amountnecessary to elicit the desired biological response. In accordance withthe present embodiments, an effective amount of a γc-antagonist is theamount necessary to provide an observable effect in at least onebiological factor for use in treating a biological condition.

“Recombinant DNA technology” or “recombinant” refers to the use oftechniques and processes for producing specific polypeptides frommicrobial (e.g., bacterial, yeast), invertebrate (insect), mammaliancells or organisms (e.g., transgenic animals or plants) that have beentransformed or transfected with cloned or synthetic DNA sequences toenable biosynthesis of heterologous peptides. Native glycosylationpattern will only be achieved with mammalian cell expression system.Prokaryotic expression systems lack the ability to add glycosylation tothe synthesized proteins. Yeast and insect cells provide a uniqueglycosylation pattern that may be different from the native pattern.

A “Nucleotide sequence” refers to a polynucleotide in the form of aseparate fragment or as a component of a larger DNA construct that hasbeen derived from DNA or RNA isolated at least once in substantiallypure form, free of contaminating endogenous materials and in a quantityor concentration enabling identification, manipulation, and recovery ofits component nucleotide sequences by standard molecular biology methods(as outlined in Current Protocols in Molecular Biology).

“Recombinant expression vector” refers to a plasmid comprising atranscriptional unit containing an assembly of (1) a genetic clement orelements that have a regulatory role in gene expression includingpromoters and enhances, (2) a structure or coding sequence that encodesthe polypeptide according to the present embodiments, and (3)appropriate transcription and translation initiation sequence and, ifdesired, termination sequences. Structural elements intended for use inyeast and mammalian system preferably include a signal sequence enablingextracellular secretion of translated polypeptides by yeast or mammalianhost cells.

“Recombinant microbial expression system” refers to a substantiallyhomogenous monoculture of suitable hot microorganisms, for example,bacteria such as E. coli, or yeast such as S. cerevisiae, that havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresidual plasmid. Generally, host cells constituting a recombinantmicrobial expression system are the progeny of a single ancestraltransformed cell. Recombinant microbial expression systems will expressheterologous polypeptides upon induction of the regulatory elementslinked to a structural nucleotide sequence to be expressed.

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

EXAMPLES Example 1 Method for Assessing the Inhibitory Activity ofγc-Antagonist Peptide

The capacity of any custom derivative peptide prepared according to thepresent embodiments for inhibiting the action of one γc-cytokine familymember is determined using mammalian cellular assays to measure theirproliferative response to the γc-cytokine family member.

For each of the six γc-cytokines, indicator cell lines: CTLL-2, a murineCD8 T cells line available from American Type Culture Collection, andPT-18, a murine mast cell line and its subclone PT-18β, is transfectedwith human IL-2β gene to make the cells responsive to IL-2 and IL-15(Tagaya et al., 1996, EMBO J. 15:4928-39), and is used to quantitativelydetermine the γc-cytokine's growth-promoting activity (See Currentprotocols in Immunology from Wiley and Sons for a methodologicalreference). The indicator cells demonstrate semi-linear dose-dependentresponse when measured by a colorimetric WST-1 assay over a range ofconcentrations (See Clontech PT3946-1 and associated user manual,incorporated herein by reference, for a detailed description of thereagents and methods). Once the appropriate doses of the cytokine thatyield the 50% and 95% maximum response from the indicator cell line isdetermined, various concentrations (ranging from 1 μM to 10 μM) of thepurified or synthesized custom derivative peptide is added to each wellcontaining the cytokine and indicator cells. The reduction in lightabsorbance at 450 nm is used as an indicator of inhibition ofcytokine-stimulated cellular proliferation. Typically, the cells arestimulated by the cytokines such that the absorbance of the wellcontaining indicator cell line and the cytokine is between 2.0 and 3.0,which is reduced to a range of 0.1 to 0.5 by the addition of inhibitorypeptides.

Example 2 BNZ-γ Peptide Specifically Inhibits the Growth-PromotingActivities of IL-9 and IL-15

Using PT-18β cells as described above, the ability of the BNZ-γ peptideto specifically inhibit the growth-promoting activity of selectγc-cytokines was determined (FIG. 3A). IL-3, a non-γc-cytokine thatsupports the growth of PT-18β cells, was used as a negative control.Briefly, PT-18β cells were incubated either with two different dilutionsof BNZ-γ peptide produced by HEK293T cells (1:20 or 1:50 dilution of theoriginal supernatant of HEK293T cells transfected with a BNZ-γexpression construct) or without BNZ-γ peptide in the presence of IL-3,IL-9, IL-15, or IL-4 (1 nM of each cytokine in the culture). Thegrowth-responses of the cells were determined 2 days after theintroduction of BNZ-γ peptide and the cytokine using the WST-1 assay.The growth-promoting activity of IL-3 (a non γc-cytokine) was notinhibited by BNZ-γ. In contrast, the activity of IL-15 and IL-9 weresignificantly (p<0.01 Student's T test) reduced by the BNZ-γ peptide.Cellular proliferation stimulated by IL-4, another γc-cytokine, was notaffected by the by the addition of BNZ-γ peptide. Results for IL-3,IL-9, IL-15, and IL-4 are shown at FIG. 3A.

In a similar assay, the murine cell line CTTL2 was used. In this assaythe cells were cultured with 0.5 nM of recombinant IL-2 in RPMI 10%fetal Calf Serum. To set up the proliferation assay, cells were washedfrom the cytokines 3 times. Cells were seeded at 1×10(5) cells per wellof a 96-well plate with final concentration of 50 μM of IL-2 or IL-15.Various concentration of BNZ-γ peptide (0.1, 1, and 10 ug/ml) was addedto each well. Cells were cultured for 20 hours and in the last 4 hours,³H-thymidine was added to the plates. Cells were harvested using a platereader. The data is shown in FIG. 3B.

Example 3 Method for Measuring Activity by Assaying 3H-thymidineIncorporation of as a Marker of Cellular Proliferation

Inhibition of γc-cytokine-induced proliferation of an indicator cellpopulation by antagonist custom derivative peptides is measured by the3H-thymidine incorporation assay. Briefly, radiolabeled thymidine (1microCi) is given to 20-50,000 cells undergoing proliferation in thepresence of cytokines. The cell-incorporated radioactivity is measuredby trapping cell-bound radioactivity to a glass-fiber filter using aconventional harvester machines (Example, Filtermate Universal Harvesterfrom Perkin-Elmer), after which the radioactivity is measured using ab-counter (Example 1450, Trilux microplate scintillation counter).

Example 4 Method for Measuring Activity by Assaying Incorporation of aCell-Tracker Dye as a Marker of Cellular Proliferation

Indicator cells are incubated in the presence of a selected γc-cytokineor in the presence of a selected γc-cytokine and a selected customderivative peptide. The cell population is then labeled in vitro using acell-tracker dye, for example, CMFDA, C2925 from Invitrogen, and thedecay of cellular green fluorescence at each cellular division ismonitored using a flow-cytometer (for example, Beckton-DickinsonFACScalibur). Typically, in response to γc-cytokine stimulation 7˜10different peaks corresponding to the number of divisions that the cellshave undergone will appear on the green fluorescence channel. Incubationof the cells with the selected γc-cytokine and antagonist customderivative peptide reduces the number of peaks to only 1 to 3, dependingon the degree of the inhibition.

Example 5 Inhibition of Intracellular Signaling by BNZ-γ and itsDerivative Antagonists

In addition to stimulating cellular proliferation, binding of theγc-cytokines to their receptors causes a diverse array of intracellularevents. (Rochman et al. 2009 Nat. Rev. Immunol. 9:480-90, Pesu et al.2005 Immunol. Rev. 203:127-142.) Immediately after the cytokine binds toits receptor, a tyrosine kinase called Jak3 (Janus-kinase 3) isrecruited to the receptor at the plasma membrane. This kinasephosphorylates the tyrosine residues of multiple proteins including theγc-subunit, STATS (Signal Transducer and Activator of Transcription 5)and subunits of the PI3 (Phosphatidylinositol 3) kinase. Among these,the phosphorylation of STATS has been implicated in many studies asbeing linked to the proliferation of cells initiated by the γc-cytokine.(Reviewed in Hennighausen and Robinson, 2008 Genes Dev. 22:711-21.) Inaccordance with these published data, whether or not the BNZ-γ peptideinhibits the tyrosine phosphorylation of STATS molecule in PT-18γ cellsstimulated by IL-15 was examined (results shown in FIG. 3C).

PT-18β cells were stimulated by IL-15 in the presence or absence ofBNZ-γ peptide. Cytoplasmic proteins were extracted from the cellsaccording to a conventional method as described in Tagaya et al. 1996EMBO J. 15:4928-39. The extracted cytoplasmic proteins were resolvedusing a standard SDS-PAGE (Sodium Dodecyl-Sulfate PolyAcrylamide GelElectrophoresis) and the phorphorylation status was confirmed by ananti-phospho-STAT5 antibody (Cell Signaling Technology, Catalog #9354,Danvers Mass.) using immunoblotting (see FIG. 3C, top panel). To confirmthat each lane represented a similar total protein load, the membranewas then stripped, and re-probed with an anti-STATS antibody (CellSignaling Technology, Catalog #9358) (see FIG. 3C, bottom panel).

These results demonstrated that tyrosine phosphorylation of STAT5, amarker of signal transduction, was induced by IL-15 in PT-18β cells, andtyrosine phosphorylation of STAT5 was markedly reduced by the BNZ-γpeptide.

Example 6 Rational Design for BNZ-γ Derivative Antagonistic Peptides

Derivative peptides are prepared based from the core sequenceD/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid) by substituting the defined amino acids of the core sequencewith amino acids having identical physico-chemical properties asdesignated in FIG. 2.

Example 7 Method of Identifying the Inhibitory Specificity ofAntagonistic Custom Derivative Peptides

The γc-cytokine inhibitory specificity of antagonistic custom derivativepeptides is determined by assaying the ability of a custom derivativepeptide to inhibit the proliferative response of a cytokine-responsivecell line to each of the 6 γc-cytokines. For example, a mouse cell line,CTLL-2, is used to determine if a candidate peptide inhibits thefunction of IL-2 and IL-15. PT-18(β) cells are used to determine if acandidate peptide inhibits the function of IL-4 and IL-9. PT-18 (7α)cells are used to determine if a candidate peptide inhibits the functionof IL-7, and PT-18(21α) cells are used to determine if a candidatepeptide inhibits the function of IL-21. PT-18(β) denotes a subclone ofPT-18 cells that exogenously express human IL-2Rβ by gene transfection(See Tagaya et al. 1996), PT-18(7α) denotes a subclone that expresseshuman IL-7Rα by gene transfection and PT-18(21Rα) cells express humanIL-21Rα.

Another alternative is to use other cell lines that respond to an arrayof cytokines. An example of this cell line in a human NK cell line NK92that is commercially available by ATCC (catalog #CRL-2407). This cellline is an IL-2 dependent cell line that responds to other cytokinesincluding IL-9, IL-7, IL-15, IL-12, IL-18, IL-21 (Gong et al. 1994Leukemia 8: 652-658, Kingemann et al., 1996, Biol Blood MarrowTransplant 2:68;75, Hodge D L et al., 2002 J. Immunol. 168:9090-8)

Example 8 Preparation of γc-Antagonist Peptides

Custom derivative γc-antagonist peptides are synthesized chemically bymanual and automated processes.

Manual synthesis: Classical liquid-phase synthesis is employed, whichinvolves coupling the carboxyl group or C-terminus of one amino acid tothe amino group or N-terminus of another. Alternatively, solid-phasepeptide synthesis (SPPS) is utilized.

Automated synthesis: Many commercial companies provide automated peptidesynthesis for a cost. These companies use various commercial peptidesynthesizers, including synthesizers provided by Applied Biosystems(ABI). Custom derivative γc-antagonist peptides are synthesized byautomated peptide synthesizers.

Example 9 Biological Production of Custom Derivative γc-AntagonistPeptides Using Recombinant Technology

A custom derivative γc-antagonist peptides is synthesized biologicallyas a pro-peptide that consists of an appropriate tagging peptide, asignal peptide, or a peptide derived from a known human protein thatenhances or stabilizes the structure of the BNZ-γ peptide and improvesits biological activity. If desired, an appropriate enzyme-cleavagesequence proceeding to the N-terminus of the peptide shall be designedto remove the tag or any part of the peptide from the final protein.

A nucleotide sequence encoding the custom derivative peptide with a stopcodon at the 3′ end is inserted into a commercial vector with a tagportion derived from thioredoxin of E. coli and a special peptidesequence that is recognized and digested by an appropriate proteolyticenzyme (for example, enterokinase) intervening between the tag portionand the nucleotide sequence encoding the custom derivative peptide andstop codon. One example of a suitable vector is the pThioHis plasmidavailable from Invitrogen, CA. Other expression vectors may be used.

EXAMPLE 10 Conjugation of the BNZ-γ and Derivative to Carrier Proteinsfor Immunization Purposes and Generation of Anti-BNZ-γ Antibody

BNZ-γ and other custom derivative peptides are used to immunize animalsto obtain polyclonal and monoclonal antibodies. Peptides are conjugatedto the N- or the C-terminus of appropriate carrier proteins (forexample, bovine serum albumin, Keyhold Limpet Hemocyanin (KLH), etc.) byconventional methods using Glutaraldehyde orm-Maleimidobenzoyl-N-Hydroxysuccinimide Ester. The conjugated peptidesin conjunction with an appropriate adjuvant are then used to immunizeanimals such as rabbits, rodents, or donkeys. The resultant antibodiesare examined for specificity using conventional methods. If theresultant antibodies react with the immunogenic peptide, they are thentested for the ability to inhibit individual γc-cytokine activityaccording to the cellular proliferation assays described in Examples1-3. Due to the composite nature of the derivative peptides it ispossible to generate a single antibody that recognizes two differentcytokines simultaneously, because of the composite nature of thesepeptides.

Example 11 Method for Large Scale Production of Custom Derivativeγc-Antagonist Peptides

Recombinant proteins are produced in large scale by the use of cell-freesystem as described elsewhere. (See Takai et al., 2010 Curr. Pharm.Biotechnol. 11(3):272-8.) Briefly, cDNAs encoding the γc-antagonistpeptide and a tag are subcloned into an appropriate vector (See Takai etal., 2010 Curr. Pharm. Biotechnol. 11(3):272-8), which is subjected toin vitro transcription, followed immediately by an in vitro translationto produce the tagged peptide. The pro-polypeptide is then purifiedusing an immobilized antibody recognizing the tagged epitope, treated bythe proteolytic enzyme and the eluate (which mostly contains the customderivative peptide of interest) is tested for purity using conventional18% Tricine-SDS-PAGE (Invitrogen) and conventional comassie staining.Should the desired purity of the peptide not be met (>98%), the mixtureis subjected to conventional HPLC (high-performance liquidchromatography) for further purification.

Example 12 Use of Custom Derivative γc-Antagonist Peptides to BlockCytokine Function in HAM/TSP

HTLV-1—associated myelopathy (HAM)/tropical spastic paraparesis (TSP) isa chronic progressive myelopathy seen in some people infected with HumanT-Lymphotropic Virus Type I (HTLV-I). Infiltration of lymphocytes in thespinal cord is associated with the immune response to HTLV-I and resultsin the release of certain cytokines. Some of these cytokines may alsodamage nerves.

Patients with HAM/TSP show an elevated state of the immune system thatis similar to that observed in autoimmune diseases (Oh et al. 2008Neurol Clin. 26:781-785). This elevated state is demonstrated by theability of HAM/TSP patient's T-cells to undergo spontaneousproliferation in an ex vivo culture for about a week in the absence ofexogenously added cytokines. The spontaneous proliferation of T-cells inHAM/TSP patients is attributed, at least partly, to autocrine/paracrineloops of IL-2, IL-9, and IL-15. It has been shown that adding blockingantibody against the IL-2 or IL-15 receptors can inhibit spontaneousT-cell proliferation in a HAM/TSP ex vivo culture system. Theseobservations, along with other data derived from ex vivo studies, haveprovided the rationale for taking two monoclonal antibodies (ananti-IL-2 receptor alpha or anti-Tac and an anti-IL-15 receptor betachain) into the clinic for treatment of HAM/TSP (Azimi et al. 2001 Proc.Natl. Acad. Sci. 98:14559-64., Azimi et al., 1999 J. Immunol163:4064-72). Anti-cytokine receptor antagonists according to theembodiments described herein, would not only be valuable as atherapeutic immuno-modulatory agent for treatment of HAM/TSP, butmodulation of immune response in HAM/TSP by anti-cytokine receptorantagonists according to the present embodiments acts proof-of-conceptfor the use of the anti-cytokine receptor antagonists according to thepresent embodiments in the treatment of other auto-immune diseases.

To demonstrate the efficacy of custom derivative γc-antagonist peptidesaccording to the embodiments described herein, we tested the ability ofBNZ-γ peptide to block immune response to HTLV-I in a spontaneous T-cellproliferation assay using a HAM/TSP ex vivo culture system.Proliferation assays were performed on HAM/TSP patient blood sampleswith and without the addition of BNZ-γ. These assays evaluated theability of BNZ-γ to block the function of cytokines, such as IL-2 andIL-15, present in the ex vivo HAM/TSP patient blood culture and preventspontaneous T-cell proliferation in these samples.

In an ex vivo spontaneous T-cell proliferation assay, PBMC from HAM/TSPpatient was cultured at 1×10(6) cells per well of a 96 well plate inRPMI-10% FCS. Increasing concentrations of BNZ-γ peptide were added toeach well. As a control, an irrelevant peptide was used in similarfashion. The cells were incubated in a 37° C. CO2 incubator for 3, 4,and 6 days. The amount of 1 uCi of ³H-thymidine was added to the cells.After an additional 6 hour incubation, cells were harvested theirproliferation rate was measured. The data for a representative HAM/TSPpatient is shown in FIG. 4A-D. As indicated in FIG. 4, BNZ-γ peptideinhibits the spontaneous proliferation of T-cells in HAM/TSP culture ata concentration of about 1 ug/ml.

Other immunological markers were additionally measured in this assay.The percentage of the viral specific CD8 cells was measured during theex vivo culture using viral protein tetramers. The population ofCD4+CD25+ cells, a marker of T-cell activation, as well as Ki67staining, a marker of T-cell proliferation, was monitored in a flowcytometry assay.

Other forms of the conjugated BNZ-γ peptide derivative can be used in asimilar future assay. They include albumin, BSA, PEG that can beconjugated to the peptide after chemical synthesis. Other biologicalforms of the BNZ-γ peptide conjugate may include regions of knownprotein entities (including but not limited to Fc region of human IgG)that are fused to the BNZ-γ peptide derivative.

Example 13 Method of Treating Adult T-cell Leukemia (ATL) in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from Adult T-cell Leukemia is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ is administered to the patientfor a period of time determined by the physician. Treatment isdetermined to be effective if patient enters remission.

Example 14 Method of Treating HAM/TSP in a Human Patient bAdministration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from HAM/TSP is identified. An effective dose,as determined by the physician, of custom derivative γc-antagonistpeptide, for example, BNZ-γ is administered to the patient for a periodof time determined by the physician. Treatment is determined to beeffective if patient's symptoms improve or if the progression of thedisease has been stopped or slowed down.

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1. An isolated or purified peptide comprising amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), wherein thepeptide can inhibit the activity of one or more γc-cytokines selectedfrom the group consisting of: IL-2, IL-4, IL-7, IL-9, IL-15 or IL-21. 2.The isolated or purified peptide of claim 1, wherein the peptideinhibits the activity of IL-9 and IL-15.
 3. The isolated or purifiedpeptide of claim 1, wherein the isolated or purified peptide furthercomprises a conjugate at the N-termini, C-termini or side residues ofthe peptide.
 4. The isolated or purified peptide of claim 3, wherein theconjugate is selected from the group consisting of: bovine serum albumin(BSA), albumin, Keyhold Limpet Hemocyanin (KLH), Fc region of IgG, abiological protein that functions as scaffold, an antibody against acell-specific antigen, a receptor, a ligand a metal ion and PolyEthylene Glycol (PEG).
 5. The isolated or purified peptide of claim 1,wherein the isolated or purified peptide further comprises a signalpeptide.
 6. The isolated or purified peptide of claim 1, wherein theisolated or purified peptide further comprises a peptide derived from aknown human protein that stabilizes the structure of the peptide andimproves its biological activity.
 7. The isolated or purified peptide ofclaim 1, wherein the isolated or purified peptide is combined in anadmixture with one or more pharmaceutically acceptable diluents,emulsifying compounds, solubilizers, adjuvants, and carriers.
 8. Theisolated or purified peptide of claim 1, wherein the isolated orpurified peptide consists essentially of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1).